Aflatoxins are secondary metabolites produced by members of the fungal genus Aspergillus. Immunosuppressive and carcinogenic activities of these toxins negatively impact human health especially in developing countries. Severity of contamination is influenced by both fungal community structure and the environment to which the crop is exposed either prior to or after harvest. In 2004, a severe episode of lethal human aflatoxicosis occurred in the Eastern Province of Kenya. Analysis of fungal community structure revealed that this event was caused by a previously unknown fungal lineage closely resembling the S strain morphotype of Aspergillus flavus. Fungal communities associated with maize produced in affected regions of Kenya were invariably dominated by the new fungal lineage and its incidence was strongly correlated with maize aflatoxin content. Analyses of fungal communities of maize grown in adjacent Kenyan provinces showed that incidences of the new lineage are limited outside the Eastern Province where the aflatoxicoses outbreaks occurred. Multi-locus phylogenetic analyses suggest the newly identified Kenyan lineage is closely related to the B and G aflatoxin producing species A. minisclerotigenes, and more distantly related to both the A. flavus S strain and an unnamed taxon with similar morphology endemic in West Africa (strain SBG). Sequence analyses of the cypA aflatoxin biosynthesis gene identified a previously unknown 2.2 kb deletion unique to the Kenyan lineage and coherent with its phylogenetic placement. A polyphasic approach was used to study aflatoxin-producing fungal communities, with emphasis on occurrence of fungi with S strain morphology, in Sub-Saharan Africa. Four phylogenetically distinct groups of fungi with S strain morphology were identified with restrictions to West Africa (strain SBG) or Central and East Africa (A. flavus S strain, A. minisclerotigenes, the new lineage). Aflatoxin production in synthetic media was a poor predictor of aflatoxin production in viable maize grain. An in vitro assay was developed to predict the aflatoxin-producing potential of fungal isolates in maize. This screen was used to identify atoxigenic isolates of A. flavus with potential value for biological control within highly toxic Aspergillus communities associated with maize production in Kenya. These atoxigenic isolates have potential value for mitigating aflatoxin contamination in Kenya.

No description available.

Mitochondria perform functions which are central to the life of most eukaryotic cells. These organelles can be considered the ultimate energy power house of a living cell. The role of mitochondria in cancer phenotype remains a fertile area of research. Several carcinogens are known to enter the mitochondria, resulting in impaired functioning and altered structure. Aflatoxin BI (AFB1) a primary type I mycotoxin elaborated by Aspergillus flavus and Aspergillus parasiticus, is carcinogenic for a wide species range. The epoxide is capable of binding to nucleic acids and proteins, resulting in induced mutations, cellular toxicity, and eventually carcinogenesis. Approximately 250 000 deaths occur annually in both China and Africa due to patients presenting with Hepatocellular Carcinoma (HCC). The causative agents being AFB1-ingestion via contaminated foods and feeds, and the Hepatitis B Virus infection. The toxin has a multifaceted mode of attack, capable of being activated to a highly reactive and carcinogenic derivative, the AFB1-8,9-epoxide, via the cytochrome P450 enzyme system of the microsomes, endoplasmic reticulum and also the mitochondria. The epoxide is capable of binding to nucleic acids and proteins, resulting in the formation of covalent adducts. The repeated occurrence of gold labelled toxin within mitochondria from hepatomas of patients presenting with HCC suggested that these organelles were direct sites of toxin binding. Despite observations that mitochondria appear as direct and perhaps preferential targets for attack by AFB1, the actual in vivo immunolocalisation and characterisation of bound AFB1 within liver mitochondria has not been reported previously. In addition the role of AFB1-protein binding within mitochondria was investigated to determine the mode of action of the toxin, within the mitochondrial system. Liver sections from rats treated with a single lethal dose of AFB1, showed distinct ultrastructural abnormalities viz. large nuclei, increased heterochromatin, and swollen mitochondria. Immunocytochemistry revealed for the first time, the selective localisation of conjugated gold labelled toxin within the mitochondria. Toxin was found in the intracristal and peripheral spaces and frequently within the mitochondrial matrix. The mitochondria isolated from treated rats revealed significant alterations and damage to the mitochondrial membranes. The cristae were also markedly swollen with the associated clearing of the mitochondrial matrix. Western blot immunoassays revealed the presence of five AFB1-bound proteins (150kDa, 50kDa, 25kDa, 18kDa, 14kDa) in the inner mitochondrial fraction of isolated mitochondria. High pressure liquid chromatography also revealed that a significant proportion (84%) of an initial dose of toxin, was absorbed by mitochondrial protein. This study is the first to show the presence of specific mitochondrial proteins involved in toxin binding. In addition, the presence of toxin within the mitochondria and the specific binding to inner mitochondrial proteins suggest that the toxin specifically targets the electron transport chain and hence effects ATP production. This study conclusively indicates that mitochondria are direct targets for attack by AFB1 during experimental carcinogenesis. Mitochondria therefore play an important role in AFB1-mediated carcinogenesis. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Aflatoxin Bi (AFBi), a highly toxic fungal metabolite (mycotoxin) of certain strains of Aspergillus, has long been known to be carcinogenic in animal species. Accumulation of epidemiological evidence led to its classification, in 1993, by the International Agency for Research on Cancer as a Group I human carcinogen. Aflatoxin Bi contaminates the food supply in most tropical and sub-tropical countries, where it is associated with increased incidence of hepatocellular carcinoma (HCC). In these countries, AFBi is also linked to kwashiorkor, jaundice, and Rey's syndrome. The biological action of AFBi is through its oxidation to AFBi-8,9 epoxide (AFBiO). This epoxide binds to macromolecules like DNA, RNA and proteins as well as amino acids to form AFBi-macromolecular adducts. Quantitation of these adducts is thought to be the most promising approach in the development of methods to measure levels of exposure to aflatoxins. Aflatoxin Bi was produced, isolated and purified using preparative thin layer chromatography (TLC). The toxin was oxidised to AFBiO using dimethyldioxirane and the UV spectra of both the AFBi and AFBiO were determined. Reaction of selected Na-acetyl amino acids (AA) with AFBiO was studied and UV spectrophotometry, TLC, high performance liquid chromatography (FfPLC) and high performance capillary electrophoresis (CE) were used to characterise the reaction products. The epoxide was also reacted with albumin and DNA. Aflatoxin Bi-albumin reaction mixture was hydrolysed and characterised by TLC. Spectrum measurement of the oxidative product of AFBi gave peaks at 266 and 367nm. Qualitative TLC and the epoxide spray reagents confirmed that epoxidation was successful. The in vivo reaction of selected Na-acetyl AA with the epoxide gave peaks between 300 and 400 nm. Naacetyl-arginine, Na-acetyl-lysine and Na-acetyl-histidine showed reaction with AFBiO with maximum wavelengths at 392, 397 and 391 nm respectively. These results strongly suggest that AFBiO is able to covalently bind to lysine, histidine and arginine in albumin. A total of twenty nine blood samples were analysed by HPLC for the presence of AFBilysyl adduct. Of the twenty nine samples, ten were from HCC patients, ten from control patients and nine from kwashiorkor patients. The results show that AFBi-lysine does occur in patients at King Edward VIII Hospital (KEH) and the highest level was detected in HCC patients followed by kwashiorkor patients. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

The aim of the present work was to investigate aflatoxin levels in various food commodities and to study its production by Aspergillus parasiticus in culture to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from afatoxins. The optimal pH for the growth of A. parasiticus and its productivity of aflatoxin B, was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production. (NH4)2HPO4 (1.55 gL-1) and NaNO2 (1.6 gL-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B, production. Zn2+ and Co2+ supported significantly both fungal growth, as well as aflatoxin B, production at the different tested concentrations. Zn2+ was effective when added to A. parasiticus growth medium at the first two days of the culture age. The other tested metal ions gave variable effects depending on the type of ion and its concentration. Water activity (a ) was an important factor controlling the growth of A. parasiticus and toxin production. The minimum aW for the fungal growth was 0.8 on both coffee beans and rice grains, while aW, of 0.70 caused complete inhibition for the growth and aflatoxin B, production. H202 is a potent inhibitor for growth of A. parasiticus and its productivity of toxins. Incubation with NaHCO3 and C6H5000Na converted aflatoxin B, to a water-soluble form which returned to aflatoxin B, by acid treatment. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B, production. Stearic acid supported the fungal growth and decreased the productivity of AFBI gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B, production. Vitamins C and D2 were also repressive particularly for aflatoxin production. The present study included determining the activities of some enzymes in relation to aflatoxin production in A. parasiticus culture during 20 days. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B, production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B, synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were correlated with the increase of aflatoxin B, production. All the tested enzymes as well as aflatoxin B, production were inhibited by either catechol or phenol.

Characterization of the 5' flanking region of rainbow trout ki-ras gene was begun with the cloning and sequencing of this region by the inverse PCR technique and dideoxynucleotide chain termination method. In total, a nucleotide sequence of 1080 bp upstream from the first coding ATG was sequenced. Although this region showed certain promoter elements, it does not share common features with other mammalian ras promoters, which lack the TATA and contain multiple GC boxes with Spl binding activities. In contrast, this region in trout ras contains typical TATA and CCAAT boxes. This structural difference of the trout ki-ras promoter from that of other mammalian ras genes may suggest that different transcriptional regulation mechanisms of the ras ger.e are used at various levels in evolution. The chromatin structure of the trout ki-ras gene was studied by probing invivo for DNase I hypersensitive sites. To overcome the difficulties of using the traditional indirect end labeling method for a single-copy gene, the technique of ligation-mediated PCR was applied. No hypersensitive sites were observed at or near the codon 12 region of the gene, either in normal (protooncogene) or tumor (oncogene) tissue from the liver. This result suggests that the local chromatin structure of trout ki-ras gene may not be an important factor for codon 12 mutations induced by genotoxins, and that changes of chromatin structure are unlikely to be promoted after tumor formation. Studies by micrococcal nuclease demonstrate that this ras gene, in the region around 12, lacks ordered nucleosome positioning or may be even free of nucleosomes. Such an irregular organization of ras oncogenic chromatin would resemble that of many other "normal", highly active eukaryotic genes. The intrinsic affinity of trout ki-ras gene for aflatoxin B₁ was determined by in vitro alkylation experiments. Exon 1 of the gene was synthesized and labeled at the 5'end of the coding strand by the PCR technique. Taking advantage of the selective cleavage of AFB1-DNA adducts by piperidine under alkali conditions, the frequency of AFB 1 attack to each guanyl site was determined by densitometric scans after the cleaved fragments were electrophoresed on sequencing gels. The results demonstrated that two guanyl sites of codon 12 had differential affinity to AFBl, the more 5' G was relatively inaccessible but the more 3' G was accessible, indicating that the sequence selectivity of AFB I may contribute to the preference of the initial adduction in vivo. / Graduation date: 1993

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on January 30, 2007) Vita. Includes bibliographical references.

Thesis (Dr. P.H.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct 31, 2007). Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed Sept. 19, 2008). Includes bibliographical references.

Thesis (M.Sc. (Pathobiology))--Mahidol University, 1983.